WO2006072954A2 - Nouveaux polynucleotides il-6 codant pour des polypeptides il-6 variants et leurs methodes d'utilisation - Google Patents

Nouveaux polynucleotides il-6 codant pour des polypeptides il-6 variants et leurs methodes d'utilisation Download PDF

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WO2006072954A2
WO2006072954A2 PCT/IL2006/000024 IL2006000024W WO2006072954A2 WO 2006072954 A2 WO2006072954 A2 WO 2006072954A2 IL 2006000024 W IL2006000024 W IL 2006000024W WO 2006072954 A2 WO2006072954 A2 WO 2006072954A2
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pea
seq
polypeptide
sequence
amino acids
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PCT/IL2006/000024
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WO2006072954A3 (fr
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Michal Ayalon-Soffer
Amir Toporik
Iris Hecht
Nir Tsabar
Zurit Levine
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Compugen Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel IL-6 variant polypeptides and polynucleotides encoding same and more particularly, to therapeutic and diagnostic methods and kits utilizing same.
  • Extracellular proteins include receptors and their corresponding ligands, play active roles in the formation, differentiation and maintenance of multicellular organisms. Any fate of an individual cell including proliferation, migration, differentiation, or interaction with other cells is typically governed by information received from distant cells and/or the immediate environment. This information is often transmitted by secreted polypeptides including but not limited to mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones, which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules are normally transferred through the cellular secretory pathway to reach their site of action at the extracellular environment.
  • Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drags available to date, including thrombolytic polypeptide sequences, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secreted proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic polynucleotide or polypeptide sequences. For example, receptor immunoadhesins can be employed as therapeutic polynucleotide or polypeptide sequences to block receptor-ligand interactions. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • Non-secreted proteins may also find application as therapeutics or diagnostics.
  • over expression of an intracellular protein (or transcript thereof) which con-elates with a disease may be used to diagnose the presence of a disease or for estimating the risk of developing a disease, by the development of probes which specifically identify the over-expressed transcript or protein.
  • the expression of the protein may be reduced using, for example, antisense or triple helix based strategies,
  • IL-6 Interleukin-6
  • IL-6 is a pleiotropic cytokine with a wide range of biological activities such as regulation of immune responses, hematopoiesis, inflammation, generation of acute-phase reactions, and oncogenesis (Naka T; et al., Research2002, 4:Suppl 3 (S233-S242)).
  • IL-6 was originally identified as an antigen-nonspecific B-cell differentiation factor in the culture supematants of mitogen- or antigen-stimulated peripheral blood mononuclear cells that induced B cells to produce immunoglobulins and was named B-cell stimulatory factor 2 (BSF-2).
  • BSF-2 B-cell stimulatory factor 2
  • IL-6 is produced by various types of lymphoid and nonlymphoid cells, such as T cells, B cells, monocytes, fibroblasts, keratinocytes, endothelial cells, mesangial cells, and several tumor cells.
  • lymphoid and nonlymphoid cells such as T cells, B cells, monocytes, fibroblasts, keratinocytes, endothelial cells, mesangial cells, and several tumor cells.
  • IL-6 production has been implicated in the pathogenesis of a variety of diseases. It has been demonstrated that chronic inflammation of the joint in rheumatoid arthritis (RA) causes IL-6 production by synovial cells, macrophages and lymphocytes in the affected synovium. Overproduction of IL-6 appears to be involved in the pathogenesis of pannus formation, angiogenesis, infiltration of mononuclear cells and destruction of cartilage and bone (Naka T; et al., Research2002.
  • RA rheumatoid arthritis
  • IL-6 is also present at very high levels in the serum and/or related tissue from patients with Crohn's disease (CD) (Yamamoto M; et al, Journal of Immunology2000, 164:9 (4878-4882)), Castleman's disease (Nishimoto N; et al., Blood2000, 95:1 (56-61)), multiple myeloma (MM) (Lauta VM, Cytokme2001.
  • CD Crohn's disease
  • MM multiple myeloma
  • IL-6R and a 13OkDa common signal transducer- gpl30, which all combine to generate a high-affinity complex of IL-6/ IL-6R/gpl30. It has pathological roles in various disease conditions, including inflammatory- mesangial proliferative glomerulonephritis, autoimmune-RA. psoriasis and malignant cancers, including but not limited to multiple myelonia/plasmacytoma and Kaposi's sarcoma.
  • YSIL6 under development of Y's Therapeutics, is a small molecule for the treatment of rheumatoid arthritis and other inflammatory disorders, and is currently in phase II clinical trials.
  • the molecule's modes of action include inhibition of TNF- ⁇ and IL-6 production in T-cells and macrophages and inhibition of T-cell migration.
  • MRA Tocilizuniab
  • Actemra a recombinant humanized MAb against human IL-6 receptor
  • Tocilizumab is under development for use in treating rheumatoid arthritis (RA), Crohn's disease, Castleman's disease and systemic lupus erythematosus (SLE),
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • An additional example is CNTO-328, a human-mouse anti-IL-6 antibody, under development by Centocor (Johnson & Johnson) for the treatment of myeloma and cachexia associated with cancer.
  • Centocor Johnson & Johnson
  • CNTO-328 is currently in phase II clinical trials.
  • ActinoDrug Pharmaceuticals 's AD-GL0002 a fungal small molecule inhibitor of IL-6 that specifically interferes with the transcription factor Stat3, thereby inhibiting the IL-6 induced Stat3/DNA pathway.
  • AD-GL0002 is being developed for the treatment of cancer and inflammation, AD-GL0002 demonstrated preclinical efficacy in an animal model of Parkinson's disease.
  • Anti-IL-6 agents also have potential in autoimmune and inflammatory diseases, such as ulcerative colitis, asthma, psoriasis, bone resoiption due to osteoporosis and RA.
  • IL-6 In view of its critical role in oncogenesis, regulation of immune response, support of hematopoiesis and generation of acute phase reaction in inflammation, there is an unmet need to develop therapies involving blocking of IL-6 function and/or its physiological effects.
  • the pathological roles of IL-6 have been clarified in various disease conditions, such as inflammatory, autoimmune, and malignant diseases. Uncontrolled IL-6 overproduction appeal's to be responsible for the clinical symptoms and abnormal laboratory findings in Rheumatoid arthritis (RA). Because of the B-cell differentiation factor activity of IL-6, overproduction of IL-6 is responsible for the increase in serum ⁇ -globulin and the emergence of rheumatoid factors.
  • IL-6 as a hepatocyte-stimulating factor causes an increase in CRP, serum amyloid A, and erythrocyte sedimentation rate and a decrease in serum albumin.
  • IL-6 as a megakaryocyte differentiation factor causes thrombocytosis. Since IL-6 in the presence of soluble IL-6R activates osteoclasts to induce bone absorption, IL-6 may be involved in the osteoporosis and destruction of bone and cartilage associated with RA. In fact, a large amount of IL-6 has been observed in both sera and synovial fluids from the affected joints of patients with RA. Blockade of the IL-6 signal may thus constitute a new therapeutic strategy for RA.
  • IL-6 was found to be involved in various diseases such as Castleman's disease, multiple myeloma/plasmacytonia, mesangial proliferative glomerulonephritis, psoriasis and Kaposi's sarcoma. Thus these diseases could be targets of IL-6 inhibitors also.
  • the present inventors have previously designed algorithms which allow for the mass prediction of new genes and gene products and for annotating these genes and gene products [see for example and without limitation LIS patent No: 6,625,545; U.S. Pat. Appl. No. 10/426,002; and PCT Application No. PCT/IL2005/000106 the teachings of all of which are incorporated herein by reference]. While applying the above-mentioned algorithms, the present inventors uncovered novel naturally occulting valiants of IL-6 gene products, which as described above, play pivotal roles in disease onset and progression. As such these variants can be used in the diagnosis and therapy of a wide range of diseases.
  • novel naturally occulting splice variants of IL-6 gene products according to the present invention can be used in the therapy and diagnosis of a wide range of variant-detectable diseases and variant- treatable diseases, which are "IL-6-related diseases " .
  • These splice variants of the present invention can be used as valuable therapeutic tools in the treatment of "IL-6- related diseases".
  • the IL-6 splice variants of the present invention can serve as antagonists (i.e., inhibitors), similarly to previously described IL-6 antagonists.
  • IL-6 variants of the present invention can optionally serve as inhibitors of IL-6 functions and/or its physiological effects.
  • IL-6-related disease(s) refers to a disease in which IL-6 activity and/or expression contribute to disease onset and/or progression, such that treating the disease may involve blocking IL-6 activity and/or expression.
  • Treatment also encompasses prevention, amelioration, elimination and/or control of the disease and/or pathological condition.
  • IL-6-related diseases include, but are not limited to, inflammatory disorders, immune disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporosis, RA, Crohn's disease.
  • the immune disorders are selected from the group consisting of ulcerative colitis, asthma.
  • the cancerous diseases are selected from the group consisting of malignant diseases-multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer.
  • the present invention envisages treatment of the above-mentioned diseases by the provision of polynucleotide or polypeptide sequences of this aspect of the present invention, which are capable of upregulating expression of the polypeptides of the present invention in a subject in need thereof, as is further described hereinbelow.
  • polynucleotide or polypeptide sequences of this aspect of the present invention and administration thereof are further described hereinbelow.
  • variant detectable disease refers to a disease in which IL-6 expression is altered as compared to the normal level.
  • variant detectable diseases include, but are not limited to, inflammatory disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporosis, RA, Crohn's disease. Castleman's disease, systemic lupus erythematosus, inflamniatory-mesangial proliferative glomerulonephritis, autoimmune-RA, psoriasis.
  • Parkinson's disease myeloproliferative disorders and cancerous diseases including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer and cachexia associated with cancer.
  • the present invention also envisages detection, diagnosis (including differential diagnosis) and/or dete ⁇ nination of prognosis of the above-mentioned diseases by the detection of polynucleotide or polypeptide sequences according to preferred embodiments of the present invention, as is further described hereinbelow.
  • detection, diagnosis including differential diagnosis
  • dete ⁇ nination of prognosis of the above-mentioned diseases by the detection of polynucleotide or polypeptide sequences according to preferred embodiments of the present invention, as is further described hereinbelow.
  • Such polynucleotide or polypeptide sequences of this aspect of the present invention and uses thereof are further described hereinbelow.
  • nucleic acid sequences of the present invention refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below.
  • oligonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.
  • disease includes any type of pathology and/or damage, including both chronic and acute damage, as well as a progress from acute to chronic damage.
  • marker in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients (subjects) having one of the herein-described diseases or conditions, as compared to a comparable sample taken from subjects who do not have one the above-described diseases or conditions.
  • a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays.
  • a polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample.
  • the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
  • a relatively low amount of up-regulation may serve as the marker, as described herein, One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
  • diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity, The "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • detecting may also optionally encompass any of the above.
  • Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be con-elated with predisposition to, or presence or absence of the disease.
  • a biological sample obtained from the subject may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.
  • the term "level” refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention. Typically the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
  • tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.
  • Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g.. brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made. Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
  • test amount of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or condition,
  • a test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • a "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker.
  • a control amount of a marker can be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition,
  • a control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • Detect refers to identifying the presence, absence or amount of the object to be detected.
  • label includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means.
  • useful labels include "P, S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample.
  • the label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
  • the label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly.
  • the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize.
  • the binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule.
  • the binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6:1 165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
  • Exemplary detectable labels include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads.
  • the marker in the sample can be detected using an indirect assay, ⁇ vherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • immunoassay is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to seminal basic protein from specific species can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example. solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g...
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • the invention provides isolated nucleic acid sequences of IL-6 variants comprising the sequences described herein. According to other embodiments, the present invention provides amino acid sequences of IL-6 variants comprising the sequences described herein.
  • any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
  • the present invention provides head, tail, bridge or edge sequence described herein.
  • the present invention provides an antibody capable of specifically binding to an epitope of an amino acid sequence of IL-6 variants comprising the sequences described herein and/or to an epitope of head, tail, bridge, edge or insertion sequence described herein.
  • the present invention provides said antibody, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
  • the invention provides a pharmaceutical composition comprising as an active ingredient any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
  • the present invention provides a biomarker capable of detecting variant-detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
  • the present invention provides a method for treating a variant-treatable disease, comprising administering a therapeutic protein, variant peptide, protein, nucleic acid sequence, antisense and/or antibody to a subject in need of treatment thereof.
  • the present invention provides a kit for detecting a variant-detectable disease, comprising a kit detecting specific expression of a splice variant as described herein.
  • the present invention provides the kit for detecting a variant-detectable disease, as above, wherein said kit comprises a NAT- based technology.
  • the present invention provides said kit, wherein said kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein. According to yet further embodiments, the present invention provides said kit, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
  • the present invention provides said kit for detecting a variant-detectable disease, as above, wherein said kit comprises an antibody as described herein. According to yet further embodiments, the present invention provides said kit, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot.
  • the present invention provides a method for detecting a variant-detectable disease, comprising detecting specific expression of a splice variant as described herein.
  • the present invention provides the method for detecting a variant-detectable disease, as above, • wherein said detecting specific expression is performed with a NAT-based technology and/or with an immunoassay.
  • the present invention provides a method for screening for variant-detectable disease, comprising detecting cells affected by a variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
  • the present invention provides a method for diagnosing a marker-detectable disease, comprising detecting cells affected by variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
  • the present invention provides a method for monitoring disease progression and/or treatment efficacy and/or relapse of ⁇ 'ariant- detectable disease, comprising detecting cells affected by variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
  • the present invention provides a method of selecting a therapy for a marker-detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay as described herein and selecting a therapy according to said detection.
  • An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) , S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) , S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) , S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) .
  • An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: S56892_PEA_l_PEA_l_node_0 (SEQ ID NO:5) , S56892_PEA_l_PEA_l_node_10 (SEQ ID NO:6)
  • S56892_PEA_l_PEA_l_node_18 S56892_PEA_l_PEA_l_node_21 (SEQ ID NO:8) , S56S92_PEAJ_PEA_l_node_3 (SEQ ID NO:9) , S56892_PEA_l_PEA_l_node_4 (SEQ ID NO: 10) , S56892_PEA_l_PEA_l_node_7 (SEQ ID NO: 1 1) , S56892_PEAJ_PEA_l_node_8 (SEQ ID NO: 12) , S56892_PEA_l_PEA_l_node_9 (SEQ ID NO: 13) , S56892_PEA_l_PEA_l_node_12 (SEQ ID NO: 14) S56892_PEA_l_PEA_l_node_13 (SEQ ID NO:
  • S56892_PEA_l_PEA_l_node_23 (SEQ ID NO:22) .
  • An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , S 56892_PEA_1_PEA_1_P 13 (SEQ ID NO:27) .
  • S56892_PEA_1_PEA_1_P8 S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , comprising a first amino acid sequence being at least about 90% homologous to
  • An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P8 comprising a polypeptide being at least about 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .
  • NE corresponding to amino acids 1 - 108 of IL6_HUMAN. which also corresponds to amino acids 1 - 108 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , and a second amino acid sequence being at least about 90% homologous to AKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALR QM corresponding to amino acids 158 - 212 of IL6_HUMAN, which also corresponds to amino acids 109 - 163 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO.25) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EA, having a structure as follows: a sequence starting from any of amino acid numbers 108-x to 108; and ending at any of amino acid numbers 109+ ((n-2) - x), in which x varies from 0 to n-2.
  • An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P11 comprising a first amino acid sequence being at least about 90% homologous to
  • MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERID KQIRYILDGISALRKETCNKSN corresponding to amino acids 1 - 76 of IL6_HUMAN, which also corresponds to amino acids 1 - 76 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) . and a second amino acid sequence being at least about 70%.
  • An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P11 comprising a polypeptide being at least about 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .
  • An isolated chimeric polypeptide encoding for an edge portion of S56892_PEA_1_PEA_1_P13 comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KE, having a structure as follows: a sequence starting from any of amino acid numbers 69-x to 69; and ending at any of amino acid numbers 70+ ((n-2) - x), in which x varies from 0 to n-2.
  • a method for treating a variant-treatable disease comprising administering a therapeutic protein, variant peptide, protein, nucleic acid sequence, antisense and/or antibody to a subject in need of treatment thereof.
  • the variant-treatable disease is cluster S56892-treatable disease and is selected from the group consisting of inflammatory disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporiosis, RA, Crohn's disease, Castleman's disease, systemic lupus erythematosus, inflammatory-mesangial proliferative glomerulonephritis, autoimmune-RA, Psoriasis, Parkinson's disease, myeloproliferative disorders and cancerous diseases, including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer and cachexia associated with cancer.
  • inflammatory disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteopo
  • nucleic acid construct comprising the isolated polynucleotide as described herein.
  • the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.
  • the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events.
  • a host cell comprising the nucleic acid construct as described herein.
  • a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as described herein and a pharmaceutically acceptable carrier or diluent.
  • a method of treating a variant- related disease in a subject comprising upregulating in the subject expression of a polypeptide as described herein, thereby treating the variant-related disease in a subject.
  • upregulating expression of said polypeptide is effected by:
  • nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
  • signalp_hmm and signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://w ⁇ vw,cbs.dtu.dk/sen ⁇ ces/SignalP/background/prediction.php) for signal peptide prediction.
  • signalpjimm and “signalp_nn” refer to two modes of operation for the program SignalP: hnim refers to Hidden Markov Model, while mi refers to neural networks. Localization was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor.
  • ProLoc computational platform [Einat Hazkani-Covo, Erez Levanon, Galit Rotman. Dan Graur and Amit Novik; (2004) Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis. Cell Biology International 2004;28(3): 171-8.], which predicts protein localization based on various parameters including, protein domains (e.g., prediction of trans- membranous regions and localization thereof within the protein), pi, protein length, amino acid composition, homology to pre-armotated proteins, recognition of sequence patterns which direct the protein to a certain organelle (such as, nuclear localization signal. NLS, mitochondria localization signal), signal peptide and anchor modeling and using unique domains from Pfam that are specific to a single compartment.
  • protein domains e.g., prediction of trans- membranous regions and localization thereof within the protein
  • pi protein length
  • amino acid composition e.g., amino acid composition
  • T - > C means that the SNP results in a change at the position given in the table from T to C.
  • M - > Q means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frameshift has occurred. A frameshift may also be indicated with a hyphen (-). A stop codon is indicated with an asterisk at the right hand side (*).
  • a comment may be found in parentheses after the above description of the SNP itself.
  • This comment may include an FTId, which is an identifier to a SwissProt entry that was created with the indicated SNP.
  • the header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence. For each given SNP, it was determined whether it was previously known by using dbSNP build 122 from NCBI, released on August 13, 2004.
  • Figure 1 shows schematic comparison of the domain structure of IL-6 variants to the known or wild-type (WT) proteins.
  • WT wild-type
  • S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25)
  • S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24)
  • S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26)
  • S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , respectively.
  • the Signal Peptide (SP) and the Helixes A, B, C and D are indicated
  • Figure 2 shows the optimized nucleotide and protein sequences for IL-6 variants according to the present invention as synthesized (including His-tag and Strep-tag sequences).
  • Figure 3 shows a schematic diagram of an exemplary construct for expressing IL-6 174 protein according to the present invention (using the nucleotide sequence shown in Figure 2).
  • Figure 4 is a Western blot of purified IL-6 174 protein according to the present invention; IL-6 174 protein itself is in lane 8, and is indicated with an arrow. Lane 10 represents lOOng of a His tagged positive control protein, and lane 1 is the molecular weight marker.
  • FIG. 5 is the PCR analysis results.
  • the high molecular weight PCR band in lane 6 represents the wild type (known) IL-6.
  • the low molecular weight PCR band in lane 7 represents the IL-6 174 variant of the present invention.
  • Figure 6 shows the results of the 250 niM Imidazole purification step. Lanes 3 and 4 contain the purified IL-6 174. Lane 1 is unpurified material and lane 2 is the molecular weight marker.
  • the present invention is of novel IL-6 variant polypeptides and polynucleotides encoding same, which can be used for the diagnosis and treatment of a wide range of diseases, such as cancer and inflammatory diseases.
  • the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein, including any oligopeptide or peptide relating to such an amino acid sequence or fragment, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges.
  • the present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
  • the present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.
  • the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides.
  • bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.
  • a "tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
  • a "head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
  • an edge portion refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein.
  • An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein.
  • a "bridge” may optionally be an edge portion as described above, but may also include a join between a head and a "known protein” portion of a variant, or a join between a tail and a "known protein” portion of a variant, or a join between an insertion and a "known protein” portion of a variant.
  • a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a valiant,
  • the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 1 1, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).
  • a bridge between two edges may optionally be described as follows: a bridge portion of CONTIG-NAME_P1 (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-NAME_P1): a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) - x) (for example), in which x varies from 0 to n-2.
  • n is any number of amino acids between 10-50 amino acids in length
  • the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1, nor 50 + ((n-2) - x) (for example) greater than the total sequence length.
  • this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
  • antibodies differentially recognize splice valiants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
  • this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
  • this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
  • this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention.
  • this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
  • the markers of the present invention can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of a marker-detectable disease.
  • these markers may be used for staging the disease in patient (for example if the disease features cancer) and/or monitoring the progression of the disease.
  • the markers of the present invention, alone or in combination can be used for detection of the source of metastasis found in anatomical places other than the originating tissue, again in the example of cancer.
  • one or more of the markers may optionally be used in combination with one or more other disease markers (other than those described herein).
  • Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease. These markers are specifically released to the bloodstream under conditions of a particular disease, and/or are otherwise expressed at a much higher level and/or specifically expressed in tissue or cells afflicted with or demonstrating the disease. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of a particular disease and/or a condition that is indicative of a higher risk for a particular disease.
  • the present invention therefore also relates to diagnostic assays for marker- detectable disease and/or an indicative condition, and methods of use of such markers for detection of marker-detectable disease and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
  • this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice valiant in the biological sample.
  • this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
  • the splice variants described herein are non-limiting examples of markers for diagnosing marker-detectable disease and/or an indicative condition.
  • Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of marker-detectable disease and/or an indicative condition, including a transition from an indicative condition to marker-detectable disease.
  • any marker according to the present invention may optionally be used alone or combination.
  • Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker.
  • such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker.
  • the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
  • Panels of markers according to the present invention optionally with one or more known marker(s)
  • the present invention is of methods, uses, devices and assays for diagnosis of a disease or condition.
  • a plurality of biomarkers may be used with the present invention.
  • the plurality of markers may optionally include a plurality of markers described herein, and/or one or more known markers.
  • the plurality of markers is preferably then correlated with the disease or condition,
  • such correlating may optionally comprise determining the concentration of each of the plurality of markers, and individually comparing each marker concentration to a threshold level.
  • the marker concentration is above or below the threshold level (depending upon the marker and/or the diagnostic test being performed)
  • the marker concentration correlates with the disease or condition.
  • a plurality of marker concentrations correlate with the disease or condition.
  • such correlating may optionally comprise determining the concentration of each of the plurality of markers, calculating a single index value based on the concentration of each of the plurality of markers, and comparing the index value to a threshold level.
  • such correlating may optionally comprise determining a temporal change in at least one of the markers, and wherein the temporal change is used in the correlating step.
  • such correlating may optionally comprise determining whether at least "X" number of the plurality of markers has a concentration outside of a predetermined range and/or above or below a threshold (as described above).
  • the value of "X" may optionally be one marker, a plurality of markers or all of the markers; alternatively or additionally, rather than including any marker in the count for "X", one or more specific markers of the plurality of markers may optionally be required to correlate with the disease or condition (according to a range and/or threshold).
  • such correlating may optionally comprise determining whether a ratio of marker concentrations for two markers is outside a range and/or above or below a threshold.
  • the ratio correlates with the disease or condition.
  • a combination of two or more these correlations may be used with a single panel and/or for correlating between a plurality of panels.
  • the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to normal subjects.
  • sensitivity relates to the number of positive (diseased) samples detected out of the total number of positive samples present; specificity relates to the number of true negative (non-diseased) samples detected out of the total number of negative samples present.
  • the method distinguishes a disease or condition with a sensitivity of at least 80% at a specificity of at least 90% when compared to normal subjects. More preferably, the method distinguishes a disease or condition with a sensitivity of at least 90% at a specificity of at least 90% when compared to normal subjects.
  • the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to subjects exhibiting symptoms that mimic disease or condition symptoms.
  • a marker panel may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a "normal" value, or a value indicating a particular outcome, A particular diagnosis/prognosis may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers are outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis.
  • diagnostic markers may be combined in a single assay or device.
  • certain markers in a panel may be commonly used to diagnose the existence of a stroke, while other members of the panel may indicate if an acute stroke has occurred, while still other members of the panel may indicate if a non-acute stroke has occulted.
  • Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different memepose(s).
  • a marker at one concentration or weighting may be used, alone or as part of a larger panel, to indicate if an acute stroke has occurred, and the same marker at a different concentration or weighting may be used, alone or as part of a larger panel, to indicate if a non-acute stroke has occurred.
  • Preferred panels comprise markers for the following purposes: diagnosis of a disease; diagnosis of disease and indication if the disease is in an acute phase and/or if an acute attack of the disease has occurred; diagnosis of disease and indication if the disease is in a non-acute phase and/or if a non-acute attack of the disease has occurred; indication whether a combination of acute and non-acute phases or attacks has occurred; diagnosis of a disease and prognosis of a subsequent adverse outcome; diagnosis of a disease and prognosis of a subsequent acute or non-acute phase or attack; disease progression (for example for cancer, such progression may include for example occurrence or recurrence of metastasis).
  • the above diagnoses may also optionally include differential diagnosis of the disease to distinguish it from other diseases, including those diseases that may feature one or more similar or identical symptoms.
  • one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s).
  • threshold level(s) of a diagnostic or prognostic indicator(s) can be established, and the level of the indicator(s) in a patient sample can simply be compared to the threshold level(s).
  • the sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test— they also depend on the definition of what constitutes an abnormal result. In practice.
  • Receiver Operating Characteristic curves are typically calculated by plotting the value of a variable versus its relative frequency in "normal” and “disease” populations, and/or by comparison of results from a subject before, during and/or after treatment. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.
  • the horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives.
  • the vertical axis of the curve represents sensitivity, which increases with the rate of true positives.
  • the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained.
  • the area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.
  • One or more markers may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such markers may be of great value in determining a particular diagnosis/prognosis.
  • particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, the present invention may utilize an evaluation of the entire marker profile by plotting ROC curves for the sensitivity of a particular panel of markers versus 1 -(specificity) for the panel at various cutoffs.
  • a profile of marker measurements from a subject is considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) that an individual has had a disease, is at risk for developing such a disease, optionally the type of disease which the individual has had or is at risk for, and so forth etc.
  • a global probability expressed either as a numeric score or as a percentage risk
  • an increase in a certain subset of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be sufficient to indicate the same or a different diagnosis/prognosis in another patient.
  • Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of particularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis.
  • markers and/or marker panels are selected to exhibit at least 70% sensitivity, more preferably at least 80% sensitivity, even more preferably at least 85% sensitivity, still more preferably at least 90% sensitivity, and most preferably at least 95% sensitivity, combined with at least 70% specificity, more preferably at least 80% specificity, even more preferably at least 85% specificity, still more preferably at least 90% specificity, and most preferably at least 95% specificity.
  • both the sensitivity and specificity are at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95%.
  • Sensitivity and/or specificity may optionally be determined as described above, with regard to the construction of ROC graphs and so forth, for example.
  • individual markers and/or combinations (panels) of markers may optionally be used for diagnosis of time of onset of a disease or condition. Such diagnosis may optionally be useful for a wide variety of conditions, preferably including those conditions with an abrupt onset.
  • determining the prognosis refers to methods by which the skilled artisan can predict the course or outcome of a condition in a patient.
  • the term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition, the chance of a given outcome may be about 3%.
  • a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance.
  • the term "about” in this context refers to +/- 1%. The skilled artisan will understand that associating a prognostic indicator with a predisposition to an adverse outcome is a statistical analysis.
  • a marker level of greater than 80 pg/niL may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to 80 pg/mL, as determined by a level of statistical significance.
  • a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events.
  • Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983.
  • Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 , and 0.0001. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter.
  • a threshold degree of change in the level of a prognostic or diagnostic indicator can be established, and the degree of change in the level of the indicator in a patient sample can simply be compared to the threshold degree of change in the level.
  • a preferred threshold change in the level for markers of the invention is about 5%, about 10%, about 15%, about 20%, about 25%.
  • a "nomogram" can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages.
  • data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers.
  • the group of subjects is divided into two sets, and preferably the first set and the second set each have an approximately equal number of subjects.
  • the first set includes subjects who have been confirmed as having a disease or, more generally, being in a first condition state.
  • this first set of patients may be those that have recently had a disease and/or a particular type of the disease.
  • the confirmation of this condition state may be made through more rigorous and/or expensive testing, preferably according to a previously defined diagnostic standard.
  • subjects in this first set will be referred to as "diseased".
  • the second set of subjects are simply those who do not fall within the first set.
  • Subjects in this second set may be "non-diseased;” that is, normal subjects.
  • subjects in this second set may be selected to exhibit one symptom or a constellation of symptoms that mimic those symptoms exhibited by the "diseased" subjects,
  • the data obtained from subjects in these sets includes levels of a plurality of markers.
  • data for the same set of markers is available for each patient.
  • This set of markers may include all candidate markers which may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required.
  • Embodiments of the methods and systems described herein may be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition.
  • the levels of each marker in the two sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required.
  • a marker often is incapable of definitively identifying a patient as either diseased or non-diseased. For example, if a patient is measured as having a marker level that falls within the overlapping region, the results of the test will be useless in diagnosing the patient.
  • An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve as described above.
  • ROC Receiveiver Operating Characteristic
  • data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to develop a panel of markers to provide a useful panel response.
  • the data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file,
  • the database or data file may contain, for example, a patient identifier such as a name or number, the levels of the various markers present, and whether the patient is diseased or non-diseased.
  • an artificial cutoff region may be initially selected for each marker.
  • the location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer, hi a preferred method, the cutoff region is initially centered about the center of the overlap region of the two sets of patients. In one embodiment, the cutoff region may simply be a cutoff point. In other embodiments, the cutoff region may have a length of greater than zero. In this regard, the cutoff region may be defined by a center value and a magnitude of length. In practice, the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range.
  • Each marker value for each patient may then be mapped to an indicator.
  • the indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis.
  • the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.
  • the relative importance of the various markers may be indicated by a weighting factor.
  • the weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer.
  • acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5.
  • the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker.
  • a panel response may be calculated for each subject in each of the two sets.
  • the panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker.
  • an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker.
  • a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state.
  • the use of an indicator which is constant on one side of the cutoff region eliminates this concern.
  • the panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient, Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker, The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response.
  • An objective function may be defined to facilitate the selection of an effective panel.
  • the objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.
  • the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function.
  • the objective function may reflect the area under the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers.
  • other features of the ROC curve may be used to define the objective function.
  • the point at which the slope of the ROC curve is equal to one may be a useful feature.
  • the point at wliich the product of sensitivity and specificity is a maximum, sometimes referred to as the "knee,” may be used.
  • the sensitivity at the knee may be maximized.
  • the sensitivity at a predetermined specificity level may be used to define the objective function.
  • Other embodiments may use the specificity at a predetermined sensitivity level may be used.
  • combinations of two or more of these ROC-curve features may be used. It is possible that one of the markers in the panel is specific to the disease or condition being diagnosed. When such markers are present at above or below a certain threshold, the panel response may be set to return a "positive" test result. When the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function.
  • An optimization algorithm may be used to maximize or minimize the objective function.
  • optimization algorithms are well-known to those skilled in the ait and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimunis.
  • the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables.
  • the weighting coefficient for each marker is also allowed to van,' across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.
  • the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.
  • the optimization algorithm may be provided with certain constraints as well.
  • the resulting ROC curve may be constrained to provide an area-under- curve of greater than a particular value.
  • ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets.
  • a minimum acceptable value such as 0.75
  • Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.
  • the iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function.
  • the number of iterations may be limited in the optimization process.
  • the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum.
  • the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators. In order to develop lower-cost panels which require the measurement of fewer marker levels, certain markers may be eliminated from the panel. In this regard, the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers.
  • the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker.
  • the markers with the lowest coefficients may be eliminated.
  • Individual panel response values may also be used as markers in the methods described herein.
  • a panel may be constructed from a plurality of markers, and each marker of the panel may be described by a function and a weighting factor to be applied to that marker (as determined by the methods described above), Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that particular marker to arrive at a sample value for that marker. The sample values for each marker are added together to arrive at the panel response for that particular sample to be tested.
  • the resulting panel responses may be treated as if they were just levels of another disease marker. Measures of test accuracy may be obtained as described in Fischer et al..
  • Intensive Care Med. 29: 1043-51, 2003 (hereby incorporated by reference as if fully set forth herein), and used to determine the effectiveness of a given marker or panel of markers.
  • These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas.
  • suitable tests may exhibit one or more of the following results on these various measures: at least 75% sensitivity, combined with at least 75% specificity; ROC curve area of at least 0.7, more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0,95; and/or a positive likelihood ratio (calculated as sensitivity/(l -specificity)) of at least 5, more preferably at least 10, and most preferably at least 20, and a negative likelihood ratio (calculated as (1- sensitivity)/specificity) of less than or equal to 0.3, more preferably less than or equal to 0.2, and most preferably less than or equal to 0.1.
  • a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof may be featured as a biomarker for detecting marker-detectable disease and/or an indicative condition, such that a biomarker may optionally comprise any of the above.
  • the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein.
  • Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges.
  • the present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
  • the present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.
  • Non-limiting examples of methods or assays are described below.
  • the present invention also relates to kits based upon such diagnostic methods or assays.
  • nucleic acid sequences described hereinabove encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
  • the present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewith, sequences homologous thereto
  • sequences encoding similar polypeptides with different codon usage sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
  • the present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.
  • the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.
  • the present invention provides isolated polynucleotides each encoding a polypeptide which is at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, %, at least 85 %, %, at least 90 %, at least 95 % or more, say 100 % identical to a polypeptide sequence listed in the Examples section or sequence listing, as determined using the LALIGN software of EMBnet Switzerland (http://www.ch.embnet.org/index.html) using default parameters.
  • a “nucleic acid fragment” or an “oligonucleotide” or a “polynucleotide” are used herein interchangeably to refer to a polymer of nucleic acids.
  • a polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent
  • genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
  • composite polynucleotide sequence refers to a sequence, which is composed of genomic and cDNA sequences.
  • a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween.
  • the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
  • Preferred embodiments of the present invention encompass oligonucleotide probes.
  • an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
  • Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.
  • Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases.
  • the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.
  • oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
  • Preferably used oligonucleotides are those modified at one or more of the backbone, intemucleoside linkages or bases, as is broadly described hereinunder.
  • oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters. methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these . , and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2', Various salts, mixed salts and free acid forms can also be used.
  • modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos.
  • oligonucleotides which can be used according to the present invention, are those modified in both sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target.
  • An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference.
  • Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.
  • Oligonucleotides of the present invention may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), A- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted ura
  • Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-niethylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammoniuni l ⁇ -di-O-hexadecyl-rac-glycero-S-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a
  • oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.
  • a nucleic acid construct (or an "expression vector") according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element.
  • cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed.
  • cell type- specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al, (1987) Genes Dev. 1 :268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473- 5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom, Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types.
  • enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation.
  • Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.
  • the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that cany the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types, Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon. If a eukaryotic ieplicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • the nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication.
  • the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice.
  • the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a vims or an artificial chromosome.
  • suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
  • retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter.
  • Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
  • Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • bone marrow cells can be targeted using the human T cell leukemia vims type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovims Autographa californica nucleopolyhedrovirus (AcMNPV) as described in Liang CY et al,, 2004 (Arch Virol. 149: 51-60).
  • Recombinant viral vectors are useful for in vivo expression of the polynucleotide sequence of the present invention since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • nucleic acid construct according to the present invention further comprises positive and negative selection markers for selecting for homologous recombination events as is known in the art.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • the expression construct of the present invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide.
  • sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide can be engineered.
  • Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein, Where a cleavage site is engineered between the Met moiety and the heterologous protein, the Met moiety can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].
  • an appropriate enzyme or agent that disrupts the cleavage site
  • prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention.
  • host-expression systems include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasniid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant vims expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid. containing the coding sequence.
  • Mammalian expression systems can also be used to express the polypeptides of the present invention.
  • bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
  • vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447.
  • vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of the coding sequence can be driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 3:17-311] can be used.
  • plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al. (1986) MoI. Cell. Biol. 6:559-565] can be used.
  • These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421- 463.
  • insects and mammalian host cell systems which are well known in the art and are further described hereinbelow can also be used by the present invention.
  • polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-limiting examples of probes according to the present invention were previously described).
  • Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA). and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.
  • Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15. 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.
  • the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
  • Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32P labeled probe, at 65 0 C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65 0 C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32P labeled probe, at 65 0 C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 0 C.
  • hybridization of short nucleic acids can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency;
  • hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 mg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature.
  • the detection of hybrid duplexes can be carried out by a number of methods.
  • hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
  • labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • a label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
  • Probes can be labeled according to numerous well known methods.
  • Non- limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.
  • Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
  • Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
  • oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
  • biotinylated dNTPs or rNTP or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs)
  • streptavidin e.g., phycoerythrin-conjugated streptavidin
  • fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
  • samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
  • Probes can be labeled according to numerous well known methods.
  • radioactive nucleotides can be incorporated into probes of the invention by several methods.
  • Non-limiting examples of radioactive labels include
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
  • Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like, Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA. Amino acid sequences and peptides
  • polypeptide polypeptide
  • peptide protein
  • polypeptide products can be biochemically synthesized such as by employing Standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis.
  • peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co, N. Y.], after which their composition can be confirmed via amino acid sequencing. In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J.
  • the present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein.
  • the present invention also encompasses homologues of these polypeptides, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low- complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50.
  • NCBI National Center of Biotechnology Information
  • the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
  • peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified. Further details in this respect are provided hereinunder.
  • Natural aromatic amino acids Tip, Tyr and Phe, may be substituted for synthetic non-natural acid such as Phenylglycine, TIC. naphthylelanine (NoI), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-niethyl-Tyr.
  • NoI naphthylelanine
  • ring- methylated derivatives of Phe ring- methylated derivatives of Phe
  • halogenated derivatives of Phe o-niethyl-Tyr.
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • the peptides of the present invention are preferably utilized in therapeutics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • the peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short ⁇ i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (L e. , not encoded by a nucleic acid sequence) and therefore involves different chemistry,
  • Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.
  • the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al.. (1987) Methods in Enzymol. 153:516- 544, Studier et al. (1990) Methods in Enzymol. 185:60-89 . . Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 10-157-311, Coruzzi et al. (1984) EMBO J. 3: 1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) MoI.
  • Antibody refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
  • the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsiloii and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g. . .
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHl, CH2 and CH3, but does not include the heavy chain variable region.
  • Fab fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain: two Fab' fragments are obtained per antibody molecule
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • Step 1 Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells
  • Producing mAb requires immunizing an animal, usually a mouse, by injection of an antigen X to stimulate the production of antibodies targeted against X.
  • Antigen X can be the whole protein or any sequence thereof that gives rise to a determinant.
  • optionally and preferably such antigens may include but are not limited to any variant described herein or a portion thereof, including but not limited to any head, tail, bridge or unique insertion, or a bridge to such head, tail or unique insertion, or any other epitope described herein according to the present invention.
  • Injection of peptides requires peptide design (with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability) and synthesis.
  • the antigen is optionally and preferably prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes optionally used as immunogens. Other immunogens or adjuvants may also optionally be used. In general, mice are immunized every 2-3 weeks but the immunization protocols are heterogeneous. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.
  • mice After several weeks of immunization, blood samples are optionally and preferably obtained from mice for measurement of serum antibodies, Several techniques have been developed for collection of small volumes of blood from mice
  • Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and/or immunoassays for example (for example a Western blot may optionally be used). If the antibody titer is high, cell fusion can optionally be performed. If the titer is too low, mice can optionally be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneal Iy or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.
  • ELISA enzyme-linked immunosorbent assay
  • Fusing antibody-producing spleen cells which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth.
  • Myeloma cells are immortalized cells that are optionally and preferably cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion.
  • the selection growth medium contains the inhibitor aminopterin, which blocks synthetic pathways by which nucleotides are made. Therefore, the cells must use a bypass pathway to synthesize nucleic acids, a pathway that is defective in the myeloma cell line to which the normal antibody-producing cells are fused.
  • the antibody forming cells are isolated from the mouse's spleen and are then fused with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely.
  • a cancer cell such as cells from a myeloma
  • the resulting cell is called a hybridoma.
  • Step 4 Fusion of Myeloma Cells with Immune Spleen Cells and antibody screening Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. Alternatively, the cells are centi ⁇ fuged, the supernatant is discarded and PEG is then added. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells (Quinlan and Kennedy 1994).
  • hybridoma colonies reach a satisfactory cell count, the plates are assayed by an assay, eg ELISA or a regular immunoassay such as RIA for example, to determine which colonies are secreting antibodies to the immunogen.
  • an assay eg ELISA or a regular immunoassay such as RIA for example.
  • Cells from positive wells are isolated and expanded.
  • Conditioned medium from each colony is retested to verify the stability of the hybridomas (that is, they continue to produce antibody).
  • Step 5 Cloning of Hybridoma Cell Lines by "Limiting Dilution” or Expansion and Stabilization of Clones by Ascites Production
  • small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time.
  • Step 6 Antibody purification
  • the secreted antibodies are optionally purified, preferably by one or more column chromatography steps and/or some other purification method, including but not limited to ion exchange, affinity, hydrophobic interaction, and gel permeation chromatography. The operation of the individual chromatography step, their number and their sequence is generally tailored to the specific antibody and the specific application.
  • In vivo production may optionally be performed with ascites fluid in mice.
  • hybridoma cell lines are injected into the peritoneal cavity of mice to produce ascitic fluid (ascites) in its abdomen; this fluid contains a high concentration of antibody.
  • An exemplar ⁇ ' in vitro method involves the use of culture flasks.
  • monoclonal antibodies can optionally be produced from the hybridoma using gas permeable bags or cell culture flasks.
  • PCT Application No. WO 94/18219 and its many US equivalents, including US Patent No. 6096551, all of which are hereby incoiporated by reference as if fully set forth herein, describes methods for producing antibody libraries using universal or randomized immunoglobulin light chains, by using phage display libraries.
  • the method involves inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene for the purpose of producing light chain gene libraries for use in combination with heavy chain genes and gene libraries to produce antibody libraries of diverse and novel immunospecificities.
  • the method comprises amplifying a CDR portion of an immunoglobulin light chain gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide.
  • PCR polymerase chain reaction
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659- 62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker, These single- chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide.
  • sFv single- chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • a scFv antibody fragment is an engineered antibody derivative that includes heavy- and light chain variable regions joined by a peptide linker.
  • the minimal size of antibody molecules are those that still comprise the complete antigen binding site. ScFv antibody fragments are potentially more effective than unmodified IgG antibodies. The reduced size of 27-30 kDa permits them to penetrate tissues and solid tumors more readily.
  • Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, La ⁇ ck and Fry [Methods, 2: 106-10 (1991)].
  • the chain could be the heavy or the light chain.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al.. Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human valuable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI, Biol., 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991)].
  • the techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p, 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • transgenic animals e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al..
  • the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.
  • An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination.
  • One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.
  • Display Libraries According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-17, 15-20, 15-30 or 20-50 consecutive amino acids derived from the polypeptide sequences of the present invention. Since in therapeutic applications it is highly desirable to employ the minimal and most efficacious polypeptide regions, which still exert therapeutic function, identification of such peptide regions can be effected using various approaches, including, for example, display techniques as described herein.
  • display vehicles such as phages, viruses or bacteria
  • a “variant-treatable” disease refers to any disease that is treatable by using a splice variant of any of the therapeutic proteins according to the present invention. “Treatment” also encompasses prevention, amelioration, elimination and control of the disease and/or pathological condition. The diseases for which such valiants may be useful therapeutic agents are described in greater detail below for each of the variants.
  • a “cluster-related disease” or a “protein-related disease” refers to a disease that may be treated by a particular protein, with regard to the description of such diseases below a therapeutic protein variant according to the present invention.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic ligand, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • modulate refers to a change in the activity of at least one receptor mediated activity. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of a ligand.
  • novel therapeutic protein variants of the present invention and compositions derived therefrom ⁇ i.e., peptides, oligonucleotides) can be used to treat cluster or protein-related diseases, disorders or conditions.
  • the subject according to the present invention is a mammal, preferably a human which is diagnosed with one of the disease, disorder or conditions described hereinabove, or alternatively is predisposed to at least one type of the cluster or protein-related disease, disorder or conditions described hereinabove.
  • 'treating refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the above-described diseases, disorders or conditions.
  • Treating can be effected by specifically upregulating or alternatively downregulating the expression of at least one of the polypeptides of the present invention in the subject.
  • upregulation may be effected by administering to the subject at least one of the polypeptides of the present invention (e.g., recombinant or synthetic) or an active portion thereof, as described herein.
  • the polypeptides of the present invention e.g., recombinant or synthetic
  • administration of polypeptides is preferably confined to small peptide fragments (e.g., about 100 amino acids).
  • the polypeptide or peptide may optionally be administered in as part of a pharmaceutical composition, described in more detail below.
  • treatment of the above-described diseases according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy).
  • treatment of malignancies using the agents of the present invention may be combined with, for example, radiation therapy, antibody therapy and/or chemotherapy.
  • an upregulating method may optionally be effected by specifically upregulating the amount (optionally expression) in the subject of at least one of the polypeptides of the present invention or active portions thereof.
  • the biomolecular sequences of this aspect of the present invention may be used as valuable therapeutic tools in the treatment of diseases, disorders or conditions in which altered activity or expression of the wild-type gene product is known to contribute to disease, disorder or condition onset or progression.
  • a soluble variant thereof may be used as an antagonist which competes with the receptor for binding the ligand, to thereby terminate signaling from the receptor. Examples of such diseases are listed in the Examples section which follows.
  • polypeptides of the present invention may also have agonistic properties. These include increasing the stability of the ligand (e.g., IL-4), protection from proteolysis and modification of the pharmacokinetic properties of the ligand (i.e. , increasing the half-life of the ligand, while decreasing the clearance thereof).
  • the biomolecular sequences of this aspect of the present invention may be used to treat conditions or diseases in which the wild-type gene product plays a favorable role, for example, increasing angiogenesis in cases of diabetes or ischemia.
  • Upregulating expression of the therapeutic protein or polypeptide variants of the present invention may be effected via the administration of at least one of the exogenous polynucleotide sequences of the present invention, ligated into a nucleic acid expression construct (as described in greater detail hereinabove) designed for expression of coding sequences in eukaryotic cells (e.g., mammalian cells), as described above.
  • the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding the variants of the present invention or active portions thereof.
  • nucleic acid construct can be administered to the individual employing any suitable mode of administration including in vivo gene therapy (e.g., using viral transformation as described hereinabove).
  • the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e. , ex-vivo gene therapy).
  • Such cells can be any suitable cells, such as kidney, bone marrow, keratinocyte, lymphocyte, adult stem cells, cord blood cells, embryonic stem cells which are derived from the individual and are transfected ex vivo with an expression vector containing the polynucleotide designed to express the polypeptide of the present inevntion as described hereinabove.
  • Administration of the ex vivo transfected cells of the present invention can be effected using any suitable route such as intravenous, intra peritoneal, intra kidney, intra gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural and rectal.
  • the ex vivo transfected cells of the present invention are introduced to the individual using intravenous, intra kidney, intra gastrointestinal track and/or intra peritoneal administrations.
  • ex vivo transfected cells of the present invention can be derived from either autologous sources such as self bone marrow cells or from allogeneic sources such as bone marrow or other cells derived from non-autologous sources. Since non- autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation.
  • Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and microencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
  • microcapsules Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. MoI Biotechnol. 2001. 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylaniine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245- 51.
  • microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 ⁇ m.
  • HEMA 2-hydroxyethyl methylacrylate
  • MAA methacrylic acid
  • MMA methyl methacrylate
  • Such microcapsules can be further encapsulated with additional 2-5 ⁇ m ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).
  • microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives.
  • microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
  • the present methodology may also be effected by specifically upregulating the expression of the valiants of the present invention endogenously in the subject.
  • Agents for upregulating endogenous expression of specific splice variants of a given gene include antisense oligonucleotides, which are directed at splice sites of interest, thereby altering the splicing pattern of the gene. This approach has been successfully used for shifting the balance of expression of the two isoforms of Bcl-x [Taylor (1999) Nat. Biotechnol.
  • interleukin 5 and its receptor play a critical role as regulators of hematopoiesis and as mediators in some inflammatory diseases such as allergy and asthma.
  • Two alternatively spliced isoforms are generated from the IL-5R gene, which include ⁇ i.e., long form) or exclude ⁇ i.e., short form) exon 9.
  • the long form encodes for the intact membrane-bound receptor, while the shorter form encodes for a secreted soluble non-functional receptor.
  • Karras and co-workers were able to significantly decrease the expression of the wild type receptor and increase the expression of the shorter isoforms. Design and synthesis of oligonucleotides which can be used according to the present invention are described hereinbelow and by Sazani and KoIe (2003) Progress in Moleclular and Subcellular Biology 31 :217-239.
  • Treatment can preferably effected by agents which are capable of specifically downregulating expression (or activity) of at least one of the polypeptide variants of the present invention.
  • oligonucleotide agents such as those described in greater detail below.
  • SiRNA molecules - Small interfering RNA (siRNA) molecules can be used to down-regulate expression of the therapeutic protein variants of the present invention.
  • RNA interference is a two-step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA).
  • nt nucleotide
  • Dicer a member of the RNase III family of dsRNA-specific ribonucleases
  • the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC.
  • the active RISC targets the homologous transcript by base pairing interactions and cleaves the niRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin.
  • RNAi RNAi RNAi RNAi RNAi RNAi amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al. Nat. Rev. Gen. 2: 110-119 (2001), Shaip Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol.
  • RNAi molecules suitable for use with the present invention can be effected as follows. First, the mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem.
  • siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
  • potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm. nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
  • an appropriate genomic database e.g., human, mouse, rat etc.
  • sequence alignment software available from the NCBI server (www.ncbi.nlm. nih.gov/BLAST/).
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
  • Several target sites are preferably selected along the length of the target gene for evaluation. Target sites are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated.
  • a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • DNAzytne molecules - Another agent capable of downregulating expression of the polypeptides of the present invention is a DNAzyme molecule capable of specifically cleaving an niRNA transcript or DNA sequence of the polynucleotides of the present invention.
  • DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997;943:4262)
  • a general model (the "10-23" model) for the DNAzyme has been proposed.
  • DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
  • This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM [Curr Opin MoI Ther 4: 119-21 (2002)].
  • Target sites for DNAzymes are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated.
  • DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al , 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL,
  • Aiitisense molecules - Downregulation of the polynucleotides of the present invention can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an niRNA transcript encoding the polypeptide variants of the present invention.
  • antisense refers to any composition containing nucleotide sequences, which are complementary to a specific DNA or RNA sequence.
  • the term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
  • Antisense molecules also include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand.
  • Antisense oligonucleotides are also used for modulation of alternative splicing in vivo and for diagnostics in vivo and in vitro (Khelifi C. et al., 2002, Current Pharmaceutical Design 8:451-1466; Sazani, P., and KoIe. R. Progress in Molecular and Cellular Biology, 2003, 31 :217-239).
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated niRNA within cells in a way which inhibits translation thereof.
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin MoI Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin MoI Ther 1 :297-306 (1999)].
  • Target sites for antisense molecules are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated.
  • Ribozymes Another agent capable of downregulating expression of the polypeptides of the present invention is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the polypeptide valiants of the present invention. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)].
  • ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials.
  • ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
  • HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).
  • TFOs triplex forming oligonuclotides
  • the triplex-forming oligonucleotide has the sequence correspondence: oligo 3'--A G G T duplex 5'-A G C T duplex 3'-T C G A
  • triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
  • Transfection of cells for example, via cationic liposomes
  • TFOs Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression.
  • Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res.
  • TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003:112:487-94).
  • Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.
  • down regulation of the polypeptide variants of the present invention may be achieved at the polypeptide level using downregulating agents such as antibodies or antibody fragments capabale of specifically binding the polypeptides of the present invention and inhibiting the activity thereof (i.e., neutralizing antibodies).
  • downregulating agents such as antibodies or antibody fragments capabale of specifically binding the polypeptides of the present invention and inhibiting the activity thereof (i.e., neutralizing antibodies).
  • Such antibodies can be directed for example, to the heterodimerizing domain on the variant, or to a putative ligand binding domain. Further description of antibodies and methods of generating same is provided below,
  • down regulation of the polypeptide variants of the present invention may be achieved using small, unique peptide sequences (e.g., of about 50- 100 amino acids) which are capable of specifically binding to their target molecules (e.g., a receptor subunit) and thus prevent endogenous subunit assembly or association and therefore antagonize the receptor activity.
  • target molecules e.g., a receptor subunit
  • Such peptides can be natural or synthetic peptides which are derived from the polypeptide of the present invention.
  • Pharmaceutical Compositions And Delivery Thereof The present invention features a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent according to the present invention, which is preferably a therapeutic protein variant as described herein.
  • the therapeutic agent could be an antibody or an oligonucleotide that specifically recognizes and binds to the therapeutic protein valiant, but not to the corresponding full length known protein.
  • the pharmaceutical composition of the present invention includes a therapeutically effective amount of at least an active portion of a therapeutic protein variant polypeptide.
  • composition according to the present invention is preferably used for the treatment of cluster or protein-related disease, disorder or condition,
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc, Preferably, the mammal is human.
  • a “disorder” is any condition that would benefit from treatment with the agent according to the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein are described with regard to specific examples given herein.
  • the term "therapeutically effective amount” refers to an amount of agent according to the present invention that is effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the agent may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • TTP time to disease progression
  • RR response rate
  • the therapeutic agents of the present invention can be provided to the subject per se, or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the preparation accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols,
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • intramuscular subcutaneous and intramedullary injections
  • intrathecal direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution. Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the ait.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such earners enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose . , hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings.
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
  • concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone. carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers,
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration,
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoiOmethane, ti ⁇ chlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoiOmethane, ti ⁇ chlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form.
  • suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran,
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized, The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • a therapeutic agent according to the present invention may optionally be a molecule, which promotes a specific immunogenic response against at least one of the polypeptides of the present invention in the subject.
  • the molecule can be polypeptide variants of the present invention, a fragment derived therefrom or a nucleic acid sequence encoding thereof.
  • the agent is preferably administered with an immunostimulant in an immunogenic composiiton.
  • An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen.
  • immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes into which the compound is incoiporated (see e.g., U.S. Pat. No. 4,235,877).
  • ⁇ accine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach),” Plenum Press (NY, 1995).
  • Illustrative immunogenic compositions may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems (see below), bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the subject (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette- Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retro vims, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent vims.
  • a viral expression system e.g., vaccinia or other pox virus, retro vims, or adenovirus
  • Suitable systems are disclosed, for example, in Fisher-Hoch et al., PI ⁇ C. Natl. Acad. Sci. USA 86:317-321 , 1989; Flexner et al., Ann. N.Y Acad. Sci.
  • an immunogenic composition may comprise both a polynucleotide and a polypeptide component. Such immunogenic compositions may provide for an enhanced immune response. Any of a variety of immunostimulants may be employed in the immunogenic compositions of this invention.
  • an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2,-7, or -12, may also be used as adjuvants.
  • Cytokines such as GM-CSF or interleukin-2,-7, or -12, may also be used as adjuvants.
  • the adjuvant composition may be designed to induce an immune response predominantly of the ThI type.
  • High levels of ThI -type cytokines e.g., IFN-. gamma., TNF. alpha., IL-2 and IL-12
  • Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-IO
  • the subject will support an immune response that includes ThI- and Th2-type responses.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffinan, Ann. Rev. Immunol. 7:145-173, 1989.
  • Preferred adjuvants for use in eliciting a predominantly ThI -type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O- acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt.
  • MPL adjuvants are available from Corixa Corporation (Seattle, Wash.: see U.S. Pat. Nos. 4,436,727; 4.877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly ThI response. Such oligonucleotides are well known and are described, for example, in WO 96/02555.
  • WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.
  • Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants,
  • QS21 Amla Biopharmaceuticals Inc., Framingham, Mass.
  • an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • compositions comprise an oil-in-water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21 , 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • a delivery vehicle may be employed within the immunogenic composition of the present invention to facilitate production of an antigen-specific immune response that targets tumor cells.
  • Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs.
  • APCs antigen presenting cells
  • Such cells may be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
  • APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmernan and Levy, Ann. Rev. Med. 50:507-529, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF. alpha, to cultures of monocytes harvested from peripheral blood.
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF. alpha., CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • Dendritic cells are categorized as "immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes.
  • Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which con-elates with the high expression of Fey receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDl 1) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
  • APCs may generally be transfected with at least one polynucleotide encoding a polypeptide of the present invention, such that variant II, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to the subject, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.
  • Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with a polypeptide of the present inventio, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
  • the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule) such as described above.
  • an immunological partner that provides T cell help e.g., a carrier molecule
  • a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
  • Preferred embodiments of the present invention encompass novel naturally occurring secreted (i.e., extracellular) and non-secreted (i.e. , intracellular or membranal) variants of genes and gene products, which, as is described in the Examples section which follows, play pivotal roles in disease onset and progression. As such these variants can be used for a wide range of diagnostic and/or therapeutic uses.
  • marker in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients having or predisposed to a cluster or protein-related disease, disorder or condition as compared to a comparable sample taken from subjects who do not have a such a disease, disorder or condition.
  • one marker or combination of markers can be measured to differentiate between various types of cluster or protein-related disease, disorder or condition, and thus are useful as an aid in the accurate diagnosis of cluster or protein-related disease, disorder or condition in a patient.
  • one marker or combination of markers can be measured to differentiate between various types of lung cancers, such as small cell or non-small cell lung cancer, and further between non-small cell lung cancer types, such as adenocarcinomas, squamous cell and large cell carcinomas, and thus are useful as an aid in the accurate diagnosis of lung cancer in a patient.
  • the present methods for detecting these markers can be applied to in vitro cluster or protein-related cancers cells or in vivo animal models for cluster or protein-related cancers to assay for and identify compounds that modulate expression of these markers.
  • a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays.
  • a polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample, It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
  • the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
  • One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided below.
  • diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.”
  • the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • predisposition refers to the susceptibility to develop a disorder.
  • a subject with a predisposition to develop a disorder is more likely to develop the disorder than a non-predisposed subject.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • detecting may also optionally encompass any of the above.
  • Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease.
  • a biological sample refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, milk, blood cells, tumors, neuronal tissue, organs, and also samples of in vivo cell culture constituents, It should be noted that a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.
  • level refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.
  • the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual.
  • tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.
  • Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g.. brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
  • Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
  • test amount of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a cluster or protein-related disease, disorder or condition related cancer or other UbcHlO related disease.
  • a test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • a "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker.
  • a control amount of a marker can be the amount of a marker in a patient which does not have the cluster or protein-related disease, disorder or condition.
  • a control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • Label includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample.
  • the label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavidin.
  • the label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly.
  • the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavidin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize.
  • the binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule,
  • the binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6: 1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
  • Exemplary detectable labels include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g.. horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads
  • the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • Immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to seminal basic protein from specific species can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988). for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
  • antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
  • this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice valiant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting the interaction: wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.
  • this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
  • the detection of the splice variant nucleic acid sequences in the biological sample is effected by detecting at least one nucleic acid change within a nucleic acid material derived from the biological sample; wherein the presence of the at least one nucleic acid change correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
  • the splice variants described herein are non-limiting examples of markers for diagnosing the cluster or protein-related disease, disorder or condition.
  • Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of such a cancer, disease or pathology.
  • any marker according to the present invention may optionally be used alone or combination.
  • Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker . , for example a known marker.
  • such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker.
  • the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
  • a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof may be featured as a biomarker for detecting the cluster or protein- related disease, disorder or condiiton, such that a biomarker may optionally comprise any of the above.
  • Non-limiting examples of methods or assays are described below.
  • the present invention also relates to kits based upon such diagnostic methods or assays.
  • Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR, or variations thereof (e.g., real-time PCR, RT-PCR and in situ RT-PCR).
  • a "primer” defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non- limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1 173-1177; Lizardi et al., 1988.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • transcription-based amplification transcription-based amplification
  • NASBA Kerardi et al.
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
  • amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
  • the oligos are designed to bind to a complementary sequence under selected conditions.
  • amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid.
  • RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA.
  • the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.
  • the nucleic acid i.e. DNA or RNA
  • Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
  • the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
  • the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition.
  • antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.
  • the polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non-limiting examples of these reactions are described in greater detail below).
  • the pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 0 C 5 preferably less than 5 0 C, more preferably less than 4 0 C, most preferably less than 3 0 C, ideally between 3 0 C and 0 0 C.
  • Tm melting temperatures
  • PCR Polymerase Chain Reaction
  • the polymerase chain reaction (PCR) as described in U.S. Pat. Nos.
  • PCR can be used to directly increase the concentration of the target to an easily detectable level.
  • This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize, Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
  • the length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are the to be "PCR-aniplified.”
  • LCR Ligase Chain Reaction
  • LAR Ligase Amplification Reaction
  • LCR LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. W09001069 Al (1990).
  • the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal, The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
  • the self-sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest.
  • the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest.
  • 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
  • Q-Beta (Q ⁇ ) Replicase In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase, A previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step.
  • available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
  • a successful diagnostic method must be very specific.
  • a straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Q ⁇ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature ⁇ i.e., > 55 degrees C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.
  • the basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle.
  • reaction conditions reduce the mean efficiency to 85 %, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material.
  • a PCR running at 85 % efficiency will yield only 21 % as much final product, compared to a reaction running at 100 % efficiency.
  • a reaction that is reduced to 50 % mean efficiency will yield less than 1 % of the possible product.
  • routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield.
  • 50 % mean efficiency it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive.
  • any background products that amplify with a better mean efficiency than the intended target will become the dominant products.
  • many variables can influence the mean efficiency of PCR including target DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition, to name but a few.
  • Contamination of the reaction with exogenous DNA e.g., DNA spilled onto lab surfaces
  • cross- contamination is also a major consideration.
  • Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator.
  • the laboriousness of this process presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way.
  • LCR LCR must also be optimized to use different oligonucleotide sequences for each target sequence.
  • both methods require expensive equipment, capable of precise temperature cycling.
  • nucleic acid detection technologies such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences.
  • One method of the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer.
  • An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence.
  • This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
  • the direct detection method may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
  • CPR cycling probe reaction
  • CPR Cycling probe reaction
  • the cycling probe reaction (CPR) uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
  • Branched DNA involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.
  • labels e.g., alkaline phosphatase enzymes
  • the NAT assays of the present invention also include methods of detecting at least one nucleic acid change [e.g., a single nucleotide polymorphism (SNP] in the biological sample of the present invention.
  • at least one nucleic acid change e.g., a single nucleotide polymorphism (SNP] in the biological sample of the present invention.
  • SNP single nucleotide polymorphism
  • nucleic acid segments for mutations or nucleic acid changes.
  • One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest.
  • amplified material e.g., PCR reaction products
  • a given segment of nucleic acid may be characterized on several other levels.
  • the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel.
  • a more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map.
  • the presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be detennined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.
  • Restriction fragment length polymorphism For detection of single- base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
  • RFLP restriction fragment length polymorphism
  • MCC Mismatch Chemical Cleavage
  • RFLP analysis suffers from low sensitivity and requires a large amount of sample.
  • RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease.
  • the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.
  • Allele specific oligonucleotide can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis-match.
  • Hybridization with radioactively labeled allelic specific oligonucleotides also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles.
  • the ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.
  • DGGE Denaturing Gradient Gel Electrophoresis
  • the fragments to be analyzed are "clamped” at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands.
  • the attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature. Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNAiRNA duplexes.
  • TGGE temperature gradient gel electrophoresis
  • the complementary strands assume sufficiently different structures that one strand may be resolved from the other, Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
  • the SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non- denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
  • Dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations.
  • the ddF technique combines components of Sanger dideoxy sequencing with SSCP.
  • a dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis.
  • ddF is an improvement over SSCP in terms of increased sensitivity
  • ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
  • Reverse dot blot This technique uses labeled sequence specific oligonucleotide probes and unlabeled nucleic acid samples. Activated primary aniine- conjugated oligonucleotides are covalently attached to carboxylated nylon membranes. After hybridization and washing, the labeled probe, or a labeled fragment of the probe, can be released using oligomer restriction, i.e., the digestion of the duplex hybrid with a restriction enzyme.
  • Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminesceiice after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film.
  • the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis, dideoxy fingerprinting, PyrosequencingTM, AcycloprimeTM, and reverse dot blot.
  • any suitable technique including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage,
  • Detection may also optionally be performed with a chip or other such device.
  • the nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group.
  • This reporter group can be a fluorescent group such as phycoerythrin.
  • the labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station.
  • a fluidics station For example, Manz et al. (1993) Adv in Chromatogr 1993; 33: 1-66 describe the fabrication of fluidics devices and particularly niicrocapillary devices, in silicon and glass substrates.
  • the chip is inserted into a scanner and patterns of hybridization are detected.
  • the hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
  • the detection of at least one nucleic acid change and/or the splice variant sequence of the present invention is effected in a biological sample containing RNA molecules using, for example.
  • RT-PCR or in situ RT-PCR RT-PCR analysis: This method uses PCR amplification of relatively rare
  • RNAs molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine.
  • a reverse transcriptase enzyme such as an MMLV-RT
  • primers such as, oligo dT, random hexamers or gene specific primers.
  • a PCR amplification reaction is carried out in a PCR machine.
  • Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semiquantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification
  • the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction.
  • the reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arctums Engineering (Mountainview, CA).
  • the GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
  • An ontology refers to the body of knowledge in a specific knowledge domain or discipline such as molecular biology, microbiology, immunology, virology, plant sciences, pharmaceutical chemistry, medicine, neurology, endocrinology, genetics, ecology, genomics, proteomics, cheminfo ⁇ natics, pharmacogenomics, bioinfomiatics, computer sciences, statistics, mathematics, chemistry, physics and artificial intelligence.
  • An ontology includes domain-specific concepts - referred to, herein, as sub- ontologies. A sub-ontology may be classified into smaller and narrower categories.
  • the ontological annotation approach is effected as follows.
  • biomolecular (/ ' e. , polynucleotide or polypeptide) sequences are computationally clustered according to a progressive homology range, thereby generating a plurality of clusters each being of a predetermined homology of the homology range,
  • Progressive homology is used to identify meaningful homologies among biomolecular sequences and to thereby assign new ontological annotations to sequences, which share requisite levels of homologies.
  • a biomolecular sequence is assigned to a specific cluster if displays a predetermined homology to at least one member of the cluster (i.e., single linkage).
  • a "progressive homology range” refers to a range of homology thresholds, which progress via predetermined increments from a low homology level (e.g. 35 %) to a high homology level (e.g. 99 %).
  • one or more ontologies are assigned to each cluster.
  • Ontologies are derived from an annotation preassociated with at least one biomolecular sequence of each cluster; and/or generated by analyzing (e.g., text- mining) at least one biomolecular sequence of each cluster thereby annotating biomolecular sequences.
  • Hierarchical annotation refers to any ontology and subontology, which can be hierarchically ordered, such as, a tissue expression hierarchy, a developmental expression hierarchy, a pathological expression hierarchy, a cellular expression hierarchy, an intracellular expression hierarchy, a taxonomical hierarchy, a functional hierarchy and so forth.
  • a dendrogram representing the hierarchy of interest is computationally constructed.
  • a "dendrogram” refers to a branching diagram containing multiple nodes and representing a hierarchy of categories based on degree of similarity or number of shared characteristics.
  • Each of the multiple nodes of the dendrogram is annotated by at least one keyword describing the node, and enabling literature and database text mining, such as by using publicly available text mining software,
  • a list of keywords can be obtained from the GO Consortium (www.geneontlogy.org). However, measures are taken to include as many keywords, and to include keywords which might be out of date.
  • tissue annotation a hierarchy is built using all available tissue/libraries sources available in the GenBank, while considering the following parameters: ignoring GenBank synonyms, building anatomical hierarchies, enabling flexible distinction between tissue types (normal versus pathology) and tissue classification levels (organs, systems, cell types, etc.).
  • each of the biomolecular sequences is assigned to at least one specific node of the dendrogram.
  • the biomolecular sequences can be annotated biomolecular sequences, unannotated biomolecular sequences or partially annotated biomolecular sequences.
  • Annotated biomolecular sequences can be retrieved from pre-existing annotated databases as described hereinabove. For example, in GenBank. relevant armotational information is provided in the definition and keyword fields. In this case, classification of the annotated biomolecular sequences to the dendrogram nodes is directly effected. A search for suitable annotated biomolecular sequences is performed using a set of keywords which are designed to classify the biomolecular sequences to the hierarchy (i.e.. same keywords that populate the dendrogram).
  • each of the assigned biomolecular sequences is recursively classified to nodes hierarchically higher than the specific nodes, such that the root node of the dendrogram encompasses the full biomolecular sequence set, which can be classified according to a certain hierarchy, while the offspring of any node represent a partitioning of the parent set.
  • a biomolecular sequence found to be specifically expressed in "rhabdomyosarcoma” will be classified also to a higher hierarchy level, which is “sarcoma”, and then to "Mesenchymal cell tumors” and finally to a highest hierarchy level “Tumor”.
  • a sequence found to be differentially expressed in endometrium cells will be classified also to a higher hierarchy level, which is "uterus”, and then to "women genital system” and to “genital system” and finally to a highest hierarchy level “genitourinary system”.
  • the retrieval can be performed according to each one of the requested levels.
  • Annotating gene expression according to relative abundance Spatial and temporal gene annotations are also assigned by comparing relative abundance in libraries of different origins. This approach can be used to find genes, which are differentially expressed in tissues, pathologies and different developmental stages. In principal, the presentation of a contigue in at least two tissues of interest is determined and significant over or under representation of the contigue in one of the at least two tissues is assessed to identify differential expression. Significant over or under representation is analyzed by statistical pairing. Annotating spatial and temporal expression can also be effected on splice variants. This is effected as follows. First, a contigue which includes exonal sequence presentation of the at least two splice variants of the gene of interest is obtained.
  • This contigue is assembled from a plurality of expressed sequences; Then, at least one contigue sequence region, unique to a portion (i.e., at least one and not all) of the at least two splice variants of the gene of interest, is identified. Identification of such unique sequence region is effected using computer alignment software. Finally, the number of the plurality of expressed sequences in the tissue having the at least one contigue sequence region is compared with the number of the plurality of expressed sequences not-having the at least one contigue sequence region, to thereby compare the expression level of the at least two splice variants of the gene of interest in the tissue.
  • Identifying gene products by interspecies sequence comparison The present inventors have designed and configured a method of predicting gene expression products based on interspecies sequence comparison. Specifically, the method is based on the identification of conserved alternatively spliced exons for which there might be no supportive expression data.
  • spliced exons have unique characteristics differentiating them from constitutively spliced ones.
  • machine-learning techniques a combination of such characteristics was elucidated that defines alternatively spliced exons with very high probability, Any human exon having this combination of characteristics is therefore predicted to be alternatively spliced.
  • the present inventors were able to detect putative splice variants that are not supported by human ESTs. The method is effected as follows.
  • alternatively spliced exons of a gene of interest are identified by scoring exon sequences of the gene of interest according to at least one sequence parameter as follows: (i) exon length - conserved alternatively spliced exons are relatively shorter than constitutively spliced ones; (ii) division by 3 - alternatively spliced exons are cassette exons that are sometimes inserted and sometimes skipped; Since alternatively spliced exons frequently contain sequences that regulate their splicing important parameters for scoring alternatively spliced exons include (iii) conservation level to a non-human ortholohgous sequence; (iv) length of conserved intron sequences upstream of each of the exon sequences; (v) length of conserved intron sequences downstream of each of the exon sequences; (vi) conservation level of the intron sequences upstream of each of the exon sequences; and (vii) conservation level of the intron sequences downstream of each of the exon sequences.
  • Exon sequences scoring above a predetermined threshold represent alternatively spliced exons of the gene of interest. Once alternatively spliced exons are identified, the chromosomal location of each of the alternatively spliced exons is analyzed with respect to coding sequence of the gene of interest to thereby predict expression products of the gene of interest. When performed along with computerized means, mass prediction of gene products can be effected. In addition, for identifying new gene products by interspecies sequence comparison, the expressed sequences derived from non-human species can be used for new human splice variants prediction. EXAMPLE 2
  • Cluster S56892 features 4 transcript(s) and 18 segment(s) of interest, the names for which are given in Tables 2 and 3, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 4.
  • S56892_PEA_1_PEA_1_P P 174 also referred to S56892_PEA_1_PEA_1 . 13 (SEQ ID NO:27) herein as IL-6 174) T14 (SEQ ID NO:4)
  • sequences are variants of the known protein Interleukin-6 precursor (SEQ ID NO:23) (SwissProt accession identifier IL6_HUMAN; known also according to the synonyms IL-6; B-cell stimulatory factor 2; BSF-2; Interferon beta-2; Hybridoma growth factor; CTL differentiation factor; CDF). referred to herein as the previously known protein.
  • Protein Interleukin-6 precursor (SEQ ID NO:23) is known or believed to have the following function(s): IL-6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig- secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation, in hepatocytes it induces acute phase reactants.
  • the sequence for protein Interleukin-6 precursor (SEQ ID NO:23) is given at the end of the application, as "Interleukin-6 precursor (SEQ ID NO:23) amino acid sequence".
  • Interleukin-6 precursor (SEQ ID NO:23) amino acid sequence is given at the end of the application, as "Interleukin-6 precursor (SEQ ID NO:23) amino acid sequence”.
  • Known polymorphisms for this sequence are as shown in Table 5,
  • Protein Interleukin-6 precursor (SEQ ID NO:23) localization is believed to be Secreted.
  • the previously known protein also has the following indication(s) and/or potential therapeutic use(s): Chemotherapy-induced injury; Cancer, sarcoma, Kaposi's; Cancer, myeloma; Chemotherapy-induced injury, bone marrow, thrombocytopenia; Thrombocytopenia; Infection, HIV/AIDS; Chemotherapy- induced injury, bone marrow, neutropenia; Cancer, breast; Cancer, colorectal; Cancer, leukaemia, acute myelogenous; Cancer, melanoma; Myelodysplastic syndrome; Hepatic dysfunction.
  • the cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication: Radio/chemoprotective; Anticancer; Cytokine; Haematological; Anti- inflammatory; Antianaemic; Antiviral, interferon; Anabolic; Hepatoprotective; Antiarthritic, immunological.
  • the following GO Annotation(s) apply to the previously known protein.
  • the following annotation(s) were found: skeletal development; acute-phase response; humoral defense mechanism; cell surface receptor linked signal transduction; cell-cell signaling; developmental processes; cell proliferation; positive control of cell proliferation; negative control of cell proliferation, which are annotation(s) related to Biological Process; cytokine; interleukin-6 receptor ligand, which are annotation(s) related to Molecular Function; and extracellular space, which are annotation(s) related to Cellular Component.
  • the GO assignment relies on information from one or more of the
  • Interleukin-6 is a pleiotropic cytokine with a wide range of biological activities in immune regulation, hematopoiesis, inflammation and oncogenesis. It acts through a combination of two different receptors, IL-6R and a 130IcDa common signal transducer-gpl30, to generate a high-affinity complex of IL-6/ IL-6R/gpl30. It has pathological roles in various disease conditions, including but not limited to inflanimatory-tnesaiigial proliferative glomerulonephritis, autoimmune-RA, Psoriasis, Parkinson's disease and cancers, including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma.
  • cluster S56892 features 4 transcript(s), which were listed in Table 1 above. These transcript(s) encode for protein(s) which are variant(s) of protein Interleukin-6 precursor (SEQ ID NO:23) . A description of each variant protein according to the present invention is now provided.
  • Variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) .
  • An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
  • the variant protein has the following domains, as determined by using InterPro. The domains are described in Table 7:
  • Variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) is encoded by the following transcript(s): S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) , for which the sequence(s) is/are given at the end of the application.
  • transcript S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) is shown in bold; this coding portion starts at position 458 and ends at position 1051,
  • the transcript also has the following SNPs as listed in Table 8 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO: 24) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) .
  • An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application, A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • chimeric polypeptide encoding for an edge portion of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EA, having a structure as follows: a sequence starting from any of amino acid numbers 108-x to 108; and ending at any of amino acid numbers 109+ ((n-2) - x), in which x varies from 0 to n-2.
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
  • Variant protein S56892_PEA_1_PEA_1_P9 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • SNPs Single Nucleotide Polymorphisms
  • the variant protein has the following domains, as determined by using InterPro. The domains are described in Table 11:
  • Variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) is encoded by the following transcript(s): S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) , for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) is shown in bold; this coding portion starts at position 113 and ends at position 601.
  • the transcript also has the following SNPs as listed in Table 12 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein
  • S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) sequence provides support for the deduced sequence of this variant protein according to the present invention.
  • Variant protein S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by trans ⁇ pt(s) S56892_PEA_1_PEA_1_T13 (SEQ ID NO:26) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by trans ⁇ pt(s) S56892_PEA_1_PEA_1_T13 (SEQ ID NO:26) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by trans ⁇ pt(s) S56892_PEA_1_PEA_1_T13 (SEQ ID NO:26) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by trans ⁇ pt(s) S56892_PEA_1_PEA_1_T13 (SEQ ID NO:26)
  • S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , comprising a first amino acid sequence being at least 90 % homologous to
  • S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .
  • the location of the valiant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
  • the variant protein has the following domains, as determined by using InterPro.
  • the domains are described in Table 14: Table 14 - Inter Pro domain(s)
  • Variant protein S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) is encoded by the following transcript(s): S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) , for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) is shown in bold; this coding portion starts at position 459 and ends at position 739.
  • the transcript also has the following SNPs as listed in Table 15 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) , An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , comprising a first amino acid sequence being at least 90 % homologous to
  • RIDKQIRYILDGISALRK corresponding to amino acids 1 - 69 of IL6JHUMAN, which also corresponds to amino acids 1 - 69 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , and a second amino acid sequence being at least 90 % homologous to
  • S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KE, having a structure as follows: a sequence starting from any of amino acid numbers 69-x to 69; and ending at any of amino acid numbers 70+ ((n-2) - x), in which x varies from 0 to n-2.
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
  • Variant protein S56892_PEA_1_PEA_1_P13 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 16, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) sequence provides support for the deduced sequence of this valiant protein according to the present invention).
  • SNPs Single Nucleotide Polymorphisms
  • the variant protein has the following domains, as determined by using InterPro. The domains are described in Table 18:
  • Variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) is encoded by the following transcript(s): S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) , for which the sequence(s) is/are given at the end of the application,
  • the coding portion of transcript S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) is shown in bold; this coding portion starts at position 458 and ends at position 979.
  • the transcript also has the following SNPs as listed in Table 19 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Figure 1 shows a schematic comparison of the domain structure of IL-6 valiants to various known or wild-type (WT) IL-6 proteins.
  • the known IL-6 antagonist P163 is given as S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) ; it lacks or u skips" exon 4 of the IL-6 gene.
  • the IL-6 variant P 198 according to the present invention is S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) ; it features intron 4 retention of the IL-6 gene.
  • the IL-6 variant P95 according to the present invention is
  • S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) and it contains a truncated exon 3.
  • the IL-6 variant Pl 74 according to the present invention is S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) . and it lacks or "skips" exon 3 of the
  • IL-6 gene The Signal Peptide (SP) and the Helixes A, B, C and D are indicated.
  • Matching Percent Similarity 100.00 Matching Percent Identity : 100.00 Total Percent Similarity : 100.00 Total Percent Identity : 100.00
  • Alignment segment 1 / 1 Alignment segment 1 / 1 :
  • Alignment segment 1 / 1 Alignment segment 1 / 1 :
  • Example 3 Validation of IL-6 variants
  • the expression of IL-6 variants according to the present invention was validated at the level of niRNA expression in renal cell carcinoma tissue.
  • the IL-6 174 transcript was validated using a junction forward primer (primer sequences are given below), The transcript was found in cDNA prepared from RNA extracted from RCC (renal cell carcinoma).
  • the experimental method used is as follows.
  • RNA was obtained from Ichilov. Total RNA samples were treated with DNaseI (Ambion Cat # 1906).
  • RTPCR - Purified RNA (1 ⁇ g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 ⁇ M dNTP in a total volume of 15.6 ⁇ l. The mixture was incubated for 5 min at 65 0 C and then quickly chilled on ice. Thereafter, 5 ⁇ l of 5X SuperscriptII first strand buffer (Invitrogen), 2.4 ⁇ l 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25 0 C, followed by further incubation at 42 0 C for 2 min.
  • Table 20 shows primers for the reaction and PCR conditions. Orientation for the primers is given as F (forward) or R (reverse).
  • PCR amplification and analysis cDNA (5ul), prepared as described above (RT PCR), was used as a template in
  • PCR reactions The amplification was done using AccuPower PCR PreMix (Bioneer, Korea, Cat# K2016), under the following conditions: IuI - of each primer (lOuM) plus 13ul - H 2 O were added into AccuPower PCR PreMix tube with a reaction program of 5 minutes at 94 0 C; 35 cycles of: [30 seconds at 94 0 C, 30 seconds at 55 0 C, 60 seconds at 72 0 C] and 10 minutes at 72 0 C.
  • products were analyzed on agarose gels stained with ethidium bromide and visualized with UV light. The PCR reaction yielded two bands (data not shown).
  • the forward primer in the high molecular weight PCR product representing the known wild type protein, is a junction forward primer used for the PCR of the low molecular weight product (GCCCTGAGAAAGGAGGAGAC: SEQ ID NO:40), which was not supposed to anneal to the WT transcript since this junction is not in the WT sequence: however, as in many PCR reaction, the primer did anneal and gave rise to the WT product as an artifact of the PCR reaction.
  • IL-6 174 variant was only found in RCC.
  • Other tissues/ cell lines tested by the above method include: blood; lymph nodes; fibroblasts; lymphocytes; and thymus. However, significant expression was not found in these other tissues/cell lines (data not shown). Therefore, it is believed that measurement of IL-6 174 levels, particularly overexpression in RCC, may be detected in any relevant samples such as kidney tissue, blood or any other suitable diagnostic sample and may optionally be used for diagnosis, prognosis, differential diagnosis and so forth of RCC.
  • the IL-6 174 sequence was codon optimized to boost protein expression in mammalian system.
  • the optimized gene was synthesized by Gene Art (Germany) by using their proprietary gene synthesis technology with the addition of DNA sequences encoding the StrepII and His tags at the 5' of the DNA fragment.
  • the gene synthesis technology is a proprietary robust nucleic acid manufacturing platform that makes double stranded DNA molecules. The resultant sequences are shown in Figure 2.
  • the bold part of the nucleotide sequence shows the relevant ORF (open reading frame) including the tag sequence, while the bold part of the amino acid sequence is the His tag (8 His residues- HHHHHHHH; SEQ ID NO:44) and Strep tag (Strep II tag: WSHPQFEK; SEQ ID NO:45) sequences.
  • His tag 8 His residues- HHHHHHHH; SEQ ID NO:44
  • Strep tag Strep tag (Strep II tag: WSHPQFEK; SEQ ID NO:45) sequences.
  • These protein tag sequences were added to all sequences so that the expressed protein can be more easily purified.
  • the DNA fragment was cloned into EcoRI/Notl sites (underlined portions of the nucleotide sequence shown in Figure 2) in pRIESpuro3 (Clontech, cat # PT3646- 5) and the sequence was verified.
  • Figure 3 shows a schematic diagram of the resultant construct.
  • the construct was transfected to HEK-293T cells (ATCC catalog number CRL-1 1268) as follows. One day prior to transfection, one well from a 6 well plate was plated with 500,000 cells in 2 ml DMEM. At the day of transfection, the FuGENE 6 Transfection Reagent (Roche, Cat#: 1-814-443) was warmed to ambient temperature and mixed prior to use. 6 ⁇ l of FuGENE Reagent were diluted into 100 ⁇ l DMEM (Dulbecco's modified Eagle's medium; Biological Industries, Cat#: 01- 055-1 A). Next, 2 micrograms of construct DNA were added. The contents were gently mixed and incubated at room temperature (RT) for 15 minutes.
  • DMEM Dulbecco's modified Eagle's medium
  • Biological Industries Cat#: 01- 055-1 A
  • the supernatants of the puromycin resistant cells were concentrated 16 fold with TCA (1 ml conditioned medium was concentrated into 6OuI). 25 ul of the solution was loaded on a 12% SDS-PAGE gel. Following electrophoresis, proteins on the gel were transferred to nitrocellulose membranes for 60 min at 35 V using Invitrogen's transfer buffer and X-CeIl II blot module. Following transfer, the blots were blocked with 5% skim milk in wash buffer (0.05% Tween-20 in PBS) for at least 60 min. at room temperature with shaking. Following blocking, the blots were incubated for 60 min at room temperature with a commercially available anti His antibody (Serotec, Cat.
  • Lane 10 in Figure 4 represents lOOng of a His tagged positive control protein, and lane 1 is the molecular weight marker.
  • the cells expressing IL-6 174 according to the present invention are taken from a T-80 flask containing serum supplemented medium after trypsinization, and are transferred into shake flasks containing serum free medium (EX-CELL293. JRH) supplemented with 4 mM glutamine and selection antibiotics (5 ug/ml puromycin). Cells are propagated in suspension at 37 0 C, 100-120 rpm agitation and culture volume is increased by sequential passages until the desired volume is reached to produce enough protein. Production-phase growth is carried out in suspension in shake flasks, spinner flasks or a stirred-tank bioreactor.
  • Protein purification IL-6 174 protein according to the present invention can be purified by two different approaches for affinity chromatography in sequential order.
  • the first approach uses Ni-NTA (nickel-nitrilotriacetic acid) resin, This type of chromatography is based on the interaction between a transition Ni 2+ ion immobilized on a matrix and the histidine side chains of His-tagged proteins. His-tag fusion proteins can be eluted from the matrix by adding free imidazole for example, as described below.
  • the second approach takes advantage of the Biotin-Streptavidin interaction principle, by using a streptavidin analog (streptactin) that is attached to the column resin, which interacts with the engineered tag StrepII.
  • the purification method used for the variants according to the present invention preferably uses the Strep/ ⁇ xHistidine system (double-tag) to ensure purification of recombinant proteins at high purity under standardized conditions.
  • IL-6 174 variant protein according to the present invention, carrying the 6xHistidine-tag and the Strep-tag II at the C - terminus, were efficiently expressed in mammalian cells.
  • IL-6 174 protein is initially purified using IMAC (Immobilized metal ion affinity chromatography) based on the 6xHistidine-tag-Ni-NTA interaction.
  • IMAC Immobilized metal ion affinity chromatography
  • the recombinant protein which also carries the Strep-tag II epitope
  • Strep-Tactin matrix No buffer exchange is required.
  • the recombinant protein is eluted from the Strep-Tactin matrix using desthiobiotin.
  • a more detailed description of the protocol is provided below; additional information about the resin and its use, as well as the resin itself, is available from IBA GmbH (Germany; http://www.iba-go.com). Since the IL6 174 variant protein initially did not bind the streptactin column, the IMAC column was repeated (1 ml column) with the same buffers. The elution was in 3 Imidazol steps (50, 100 and 250 mM). Most of the IL6 174 protein was eluted in 250 mM Imidazole. 20 ul of the solution was loaded on a 4-12% SDS-PAGE gel.
  • Figure 6 shows the results of the 250 mM Imidazole purification step.
  • Lanes 3 and 4 contain the purified IL-6 174.
  • Lane 1 is unpurified material and lane 2 is the molecular weight marker.
  • This gel shows that optionally the IL-6 174 valiant protein according to the present invention may be purified by using two columns that rely on the His-tag for purification, rather than a first column relying on the His-tag followed by a second column relying on the strep-tag.
  • Reagents D-Desthiobiotin; 5 g ⁇ IBA, Cat# 2-1000-005 ⁇ ; Dulbecoo's Phosphate Buffered Saline (PBS), concentrate XlO ⁇ Biological Industries, Cat # 020235A ⁇ ; Sodium Phosphate ⁇ Sigma, Cat # S7907) ; Strep-tag® regeneration buffer with HABA; 100 ml ⁇ IBA, Cat# 2-1002-100 ⁇ ; Millipore filters . .
  • Buffers Ni-NTA Binding & Wash Buffer (Buffer A): 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0; Ni-NTA Elution Buffer (Buffer B): 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0 ; Streptactin wash buffer (Buffer A): 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0; Strepactin elution buffer (Buffer D): 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, 2.5 mM desthiobiotin, pH 8.0.
  • IL-6 174 protein according to the present invention is purified by affinity chromatography using Ni-NTA resin, according to the following protocol.
  • 2L of culture is concentrated to 200 ml by ultrafiltration.
  • Imidazole is added to the sample to final concentration of 10 mM and the sup is filtered through a 0.22 um filter (Millipore).
  • the supernatant is transferred to a 250 ml centrifuge tube.
  • Four ml of Ni-NTA Superflow beads are equilibrated with 10 column volumes of WFI and 10 column volumes of Buffer A (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0). The beads are added to the filtered supernatant, and the tube is incubated overnight on a rocking platform at 4 0 C.
  • IL-6 valiants The in vitro biological activity of IL-6 valiants is assessed in a cell proliferation assay using the 7TDl cell line (DSMZ cat # ACC23).
  • 7TDl cell line DSMZ cat # ACC23
  • an IL-6-dependent cell line which originated in murine myeloma cells fused to murine B-cells and which has been used for the development of IL-6 antagonists (Manfredini et al., Peptides 2003, 24:1207-1220).
  • Sant7 a known mutein and potent antagonist of IL-6 activity (Sporeno et al.. Blood, 1996, 87: 4510-4519), serves as positive control for antagonist activity.
  • the activity of each variant is tested alone (i.e. as an agonist) or in the presence of commercial human IL-6 (i.e.
  • the cells are plated in 96- well plates, and the IL-6 174 variant or the positive control are added in different concentrations . , in the presence or absence of 50 pg/ml human IL-6. Proliferation is assessed 72 hrs later by MTT assay or by BrdU incorporation assay.
  • myeloma the variety of genetic changes and oncogene mutations that characterize myeloma may give rise to different cells, whose sensitivity to IL-6 diverges in both quantity and quality, while still maintaining their IL-6 response.
  • U266 (ATCC cat no.: TIB- 196) is a human meyeloma cell line that produces endogenous IL-6 which stimulates cell proliferation via an autocrine loop. These cells have been previously used as in-vitro model for studying IL-6 antagonists (Alberti et al., Cancer Res 2005; 65:2-5; Sporeno et al., Blood, 1996, 87: 4510-4519). Cells are maintained in RPMI, 15% FCS. For proliferation assay, the cells (105/ml) are washed and resuspended in low serum medium (1% FCS) and plated in a 96 microtiter plate in 100 ml.
  • IL-6 174 splice variant or a known antagonistic mutein, such as SANT7 (as positive control), are added to the cells. Proliferation is measured 72 hr later, using BrdU ELISA.
  • Other human multiple myeloma cell lines, such as INA-6 and XG-I may be used in cell proliferation, cell cyle analysis and/or apoptosis assays, in order to evaluate the potential of IL-6 174 variant to exert an antagonistic effect, as has been described previously (Tassone et al, Clin. Cancer Res., 2005, 11 :4251-4258; Sporeno et al., Blood, 1996, 87: 4510-4519; Petrucci et al 1999. Ann Hematol 78: 13-18).
  • B9 (DSMZ cat no.: ACC 211) is a mouse hybridoma cell line which resulted from the fusion of murine myeloma cells with spleen B cells. This cell line is totally dependent on IL-6 for growth, and thus serves as a model system for studying potential IL-6 antagonists (Alberti et al., Cancer Res 2005; Brakenhoff et al., J, Biol Chemistry, 1994, 269:86-93). B9 cells are maintained in RPMI, 10% FBS, 50 ⁇ M mercaptoethanol, 100 pg/ml human IL-6.
  • IL-6 174 splice variant and a known antagonistic mutein (as positive control) on B9 proliferation
  • the cells are washed and resuspended (2xlO 4 /ml) in RPMI containing 1% FCS and plated at 100 ⁇ l/well in a 96- well microtiter plate in IL-6 174 splice variant or positive control mutein are added from serial dilutions, in the presence or absence of 3pg/ml IL-6, in order to assess their agonistic and antagonistic activities.
  • Proliferation is measured 72 Iu- later, using BrdU incorporation or MTT assay,
  • A375 (ATCC cat no.: CRL-1619) are human malignant melanoma cells that respond to IL-6 by growth arrest. These cells have been used to analyze the activity of IL-6 antagonists (Sporeno et al., Blood 1996 87(11):4510-9; Savino, R., et al., EMBO
  • IL-6 174 splice variant is added from serial dilutions to A375 cells, in a 96-well microtiter plate containing 5000 cells/well . , in the presence or absence of human known (WT) IL-6 as an agonist. Cell survival is evaluated by BrdU incorporation or MTT assays.
  • CESS (ATCC cat no.: TIB- 190), a human myelomonocytic leukemia cell line, is used for this assay. Stimulation of CESS cells with IL-6 results in increased IgGl secretion, and has been used to assess IL-6 antagonist activity (Brakenhoff 1994,). The effect of IL-6 174 splice variant on IgGl secretion is studied by incubating these cells with serial dilutions of the splice variant or a known antagonistic mutein, such as SANT7 (as positive control), in the presence or absence of human IL-6. Levels of IgGl secretion are assessed using an ELISA, as is well known in the ait.
  • SKW6.4 (ATCC cat no.: TIB-215) is a human EBV transformed B cell line with plasmacytoid morphology. These cells respond to IL-6 stimulation by increased IgM secretion (10-30 fold) and have been used to analyze IL-6 antagonists (Shiao, et al. Leukemia and Lymphoma, 1995, 17:485-494; Peppard et al J. Biol. Chem., 1996, 271 : 7281-7284).
  • IL-6 174 splice variant The effect of IL-6 174 splice variant on IgM secretion is studied by incubating the cells (10000 cells/well in 96-well plate) with serial dilutions of the IL-6 174 splice variant or a known antagonist . , such as SANT7 (as positive control), in the presence or absence of IL-6 for a total of 3 days. IgM secretion is assessed using an ELISA as is well known in the art.
  • Blocking of IL-6 functions following a delivery of a therapeutic amount of IL- 6 variants of the present invention in the cynomolgus monkey is assessed by inhibition of two functional parameters in vitro: T-cell proliferation stimulated by phytohemaglutinin and human IL-6, and IgG production evoked by Staphylococcus aureus Cowan- 1- and human IL-6-stimulated B-lymphocytes.
  • IL-6 valiants of the present invention The in vivo effect of IL-6 valiants of the present invention on the development of collagen-induced arthritis is examined in cynomolgus monkeys (the same model as for overall assessment of the effect of IL-6 174 variant on IL-6 functionality in vivo). Inhibition of arthritis symptoms is measured following delivery of therapeutic amount of proteins of the IL-6 variants of the present invention. Inhibition of the elevation of serum CRP and fibrinogen levels, and inhibition of erythrocyte sedimentation rate (ESR) are measured as well. Furthermore, radiographic and histological examination is carried out, showing that IL-6 variant treatment suppresses joint destruction.
  • ESR erythrocyte sedimentation rate
  • IL-6 variants of the present invention are an attractive agent for the treatment of RA as they decrease the number of inflammatory cells and metalloproteinase-positive cells in the implanted tissues.
  • the IL-6 174 variant is used.
  • a subject diagnosed with RA is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Castleman's disease is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Crohn's disease is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with SLE is treated with an IL-6 valiant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • ip injection of rat anti-murine IL-6R antibody (2 mg at the time of colitis induction and 1 mg weekly for up to 8 weeks) suggests a therapeutic potential of IL-6 variants of the present invention in the treatment of colitis.
  • a subject diagnosed with colitis is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a novel murine model of human multiple myeloma in which IL-6-dependent INA-6 multiple myeloma cells are directly injected into human bone marrow implants in severe combined immunodeficient (SCID) mice (SCID-hu), is used to assess the effect of IL-6 variant treatment on inhibition of the growth of myeloma cells.
  • SCID-hu severe combined immunodeficient mice
  • the effect of in vivo drug treatments on multiple myeloma cell growth is monitored by serial determinations of serum levels of soluble IL-6 receptor (shuIL-6R), which is released by INA-6 cells and serves as a marker of tumor growth.
  • shuIL-6R soluble IL-6 receptor
  • a subject diagnosed with multiple myeloma is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Leukemia and/or Lymphoma is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with renal cell carcinoma is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • IL-6 valiants of the present invention particular IL-6 174, are potential therapeutic agents for the treatment of tumor-related cachexia.
  • a subject diagnosed with tumor-related cachexia is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease.
  • An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject.
  • the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with RA is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct.
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Castleman's disease is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct,
  • the sequences encoding one or more of the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • the physical characteristics of the subject e.g.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Crohn's disease is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct,
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with SLE is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed //; vivo from the expression construct.
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with colitis is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct.
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable vims containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Multiple Myeloma is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice valiant proteins of the present invention are expressed in vivo from the expression construct.
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with Leukemia and/or Lymphoma is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease,
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct.
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable vims containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with renal cell carcinoma is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct.
  • the sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable vims containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • the physical characteristics of the subject e.g.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment.
  • additional doses are monitored from about daily to about weekly.
  • a subject diagnosed with tumor-related cachexia is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease.
  • the IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct.
  • the sequences encoding the splice valiant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter.
  • a suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression.
  • a dose containing a particular concentration of vector is delivered by intravenous injection.
  • the subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Abstract

L'invention concerne des nouveaux polypeptides IL-6 variants et des nouveaux polynucléotides codant pour ces derniers. L'invention concerne également des méthodes et des compositions pharmaceutiques qui peuvent être utilisées pour traiter divers troubles tels que le cancer, des troubles associés au système immunologique, au système sanguin et à la peau au moyen des polypeptides et des polynucléotides de la présente invention. L'invention concerne également des méthodes et des trousses permettant de diagnostiquer, de déterminer la prédisposition et/ou le pronostic de divers troubles en utilisant, comme marqueurs diagnostiques, les nouveaux polypeptides IL-6 variants et polynucléotides selon l'invention.
PCT/IL2006/000024 2005-01-05 2006-01-05 Nouveaux polynucleotides il-6 codant pour des polypeptides il-6 variants et leurs methodes d'utilisation WO2006072954A2 (fr)

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US7939634B2 (en) 2004-01-27 2011-05-10 Compugen Ltd. Polynucleotides encoding polypeptides and methods using same
US8198414B2 (en) 2006-11-30 2012-06-12 Medimmune Limited Anti-human IL-6 antibodies
CN104906581A (zh) * 2008-06-05 2015-09-16 国立研究开发法人国立癌症研究中心 神经浸润抑制剂
US9347952B2 (en) 2005-10-03 2016-05-24 Compugen Ltd. Soluble VEGFR-1 variants for diagnosis of preeclampsia
US9725514B2 (en) 2007-01-23 2017-08-08 Shinshu University Chronic rejection inhibitor
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US10782290B2 (en) 2013-06-11 2020-09-22 National Center Of Neurology And Psychiatry Method for predicting post-therapy prognosis of relapsing-remitting multiple sclerosis (RRMS) patient, and method for determining applicability of novel therapy
US11203636B2 (en) 2017-02-01 2021-12-21 Yale University Treatment of existing left ventricular heart failure
US11384143B2 (en) 2018-01-05 2022-07-12 Novo Nordisk A/S Methods for treating IL-6 mediated inflammation without immunosuppression
US11692037B2 (en) 2017-10-20 2023-07-04 Hyogo College Of Medicine Anti-IL-6 receptor antibody-containing medicinal composition for preventing post-surgical adhesion
US11851486B2 (en) 2017-05-02 2023-12-26 National Center Of Neurology And Psychiatry Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7939634B2 (en) 2004-01-27 2011-05-10 Compugen Ltd. Polynucleotides encoding polypeptides and methods using same
US9347952B2 (en) 2005-10-03 2016-05-24 Compugen Ltd. Soluble VEGFR-1 variants for diagnosis of preeclampsia
US8198414B2 (en) 2006-11-30 2012-06-12 Medimmune Limited Anti-human IL-6 antibodies
US9005620B2 (en) 2006-11-30 2015-04-14 Medimmune Limited Compounds
US9725514B2 (en) 2007-01-23 2017-08-08 Shinshu University Chronic rejection inhibitor
US10717781B2 (en) 2008-06-05 2020-07-21 National Cancer Center Neuroinvasion inhibitor
CN104906581A (zh) * 2008-06-05 2015-09-16 国立研究开发法人国立癌症研究中心 神经浸润抑制剂
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US10782290B2 (en) 2013-06-11 2020-09-22 National Center Of Neurology And Psychiatry Method for predicting post-therapy prognosis of relapsing-remitting multiple sclerosis (RRMS) patient, and method for determining applicability of novel therapy
US11203636B2 (en) 2017-02-01 2021-12-21 Yale University Treatment of existing left ventricular heart failure
US11851486B2 (en) 2017-05-02 2023-12-26 National Center Of Neurology And Psychiatry Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils
US11692037B2 (en) 2017-10-20 2023-07-04 Hyogo College Of Medicine Anti-IL-6 receptor antibody-containing medicinal composition for preventing post-surgical adhesion
US11384143B2 (en) 2018-01-05 2022-07-12 Novo Nordisk A/S Methods for treating IL-6 mediated inflammation without immunosuppression

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